Ruth M. Kluck

Cytochrome c activation of CPP32‐like proteolysis plays a critical role in a Xenopus cell‐free apoptosis system

In a cell‐free system based on Xenopus egg extracts, Bcl‐2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase‐3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32‐like protease activity plays an essential role in this system, as the caspase inhibitor, Ac‐DEVD‐CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro‐apoptotic function, as Cu‐ and Zn‐substituted cytochrome c had strong pro‐apoptotic activity, despite being redox‐inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell‐free system, cytosol‐dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32‐like caspases, via unknown cytosolic factors.

Bax Crystal Structures Reveal How Bh3 Domains Activate Bax and Nucleate its Oligomerization to Induce Apoptosis.

In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each others surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.

Molecular biology of Bax and Bak activation and action.

Bax and Bak are two nuclear-encoded proteins present in higher eukaryotes that are able to pierce the mitochondrial outer membrane to mediate cell death by apoptosis. Thus, organelles recruited by nucleated cells to supply energy can be recruited by Bax and Bak to kill cells. The two proteins lie in wait in healthy cells where they adopt a globular α-helical structure, seemingly as monomers. Following a variety of stress signals, they convert into pore-forming proteins by changing conformation and assembling into oligomeric complexes in the mitochondrial outer membrane. Proteins from the mitochondrial intermembrane space then empty into the cytosol to activate proteases that dismantle the cell. The arrangement of Bax and Bak in membrane-bound complexes, and how the complexes porate the membrane, is far from being understood. However, recent data indicate that they first form symmetric BH3:groove dimers which can be linked via an interface between the α6-helices to form high order oligomers. Here, we review how Bax and Bak change conformation and oligomerize, as well as how oligomers might form a pore. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.

To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions.

The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.

Mechanisms by which Bak and Bax permeabilise mitochondria during apoptosis

Mitochondrial outer membrane permeabilisation (MOMP) is the point of no return in many forms of apoptotic cell death. The killing effect of MOMP is twofold; it both initiates a proteolytic cascade of pro-apoptotic enzymes and damages mitochondrial function. Accordingly, prevention of MOMP can rescue cells from death. It is clear that either Bak or Bax, which are Bcl-2 family members, are required for MOMP to occur; however, the pore complexes that are formed by Bak and Bax remain poorly defined in terms of their composition, size, number and structure, as well as the mechanism by which they are regulated by other Bcl-2 family members. We recently reported that a key step leading to Bak homo-oligomerisation following an apoptotic stimulus involves transient exposure of the Bak BH3 domain before it binds to the hydrophobic groove of another activated Bak molecule to form a novel symmetric dimer. To form the higher-order oligomers that probably constitute the apoptotic pore complex, Bak dimers then interact via regions away from the BH3 domain and groove. The BH3:groove interaction within Bak homodimers supports a general model to explain the associations between Bcl-2 family members. In this Commentary, we discuss the implications of these findings for the regulation of apoptosis by Bcl-2 family proteins.

Bak Activation for Apoptosis Involves Oligomerization of Dimers via Their α6 Helices

A pivotal step toward apoptosis is oligomerization of the Bcl-2 relative Bak. We recently reported that its oligomerization initiates by insertion of an exposed BH3 domain into the groove of another Bak monomer. We now report that the resulting BH3:groove dimers can be converted to the larger oligomers that permeabilize mitochondria by an interface between alpha6 helices. Cysteine residues placed in alpha6 could be crosslinked only after apoptotic signaling. Cysteines placed at both interfaces established that the BH3:groove dimer is symmetric and that the alpha6:alpha6 interface can link these dimers into homo-oligomers containing at least 18 Bak molecules. A putative zinc-binding site in alpha6 was not required to form the alpha6:alpha6 interface, and its mutation in full-length Bak did not affect Bak conformation, oligomerization, or function. We conclude that alpha6:alpha6 interaction occurs during Bak oligomerization and proapoptotic function, but we find no evidence that zinc binding to that interface regulates apoptosis.

Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis

The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different ‘modes’ by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane.

A Distinct Pathway of Cell-Mediated Apoptosis Initiated by Granulysin

Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Δψ) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.

Mitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak

The Bcl-2 family regulates apoptosis by controlling mitochondrial integrity. To clarify whether its prosurvival members function by sequestering their Bcl-2 homology 3 (BH3)–only ligands or their multidomain relatives Bak and Bax, we analyzed whether four prosurvival proteins differing in their ability to bind specific BH3 peptides or Bak could protect isolated mitochondria. Most BH3 peptides could induce temperature-dependent cytochrome c release, but permeabilization was prevented by Bcl-xl, Bcl-w, Mcl-1, or BHRF1. However, their protection correlated with the ability to bind Bak rather than the added BH3 peptide and could be overcome only by BH3 peptides that bind directly to the appropriate prosurvival member. Mitochondria protected by both Bcl-xl–like and Mcl-1 proteins were disrupted only by BH3 peptides that engage both. BH3-only reagents freed Bak from Bcl-xl and Mcl-1 in mitochondrial and cell lysates. The findings support a model for the control of apoptosis in which certain prosurvival proteins sequester Bak/Bax, and BH3-only proteins must neutralize all protective prosurvival proteins to allow Bak/Bax to induce mitochondrial disruption.

Determinants of Cytochrome c Pro-apoptotic Activity THE ROLE OF LYSINE 72 TRIMETHYLATION

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72,N-ε-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome cexpressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.